This invention relates to novel inhibitors of the Nuclear factor kappa B (NF-xcexaB) activating pathway useful in the treatment of NF-xcexaB related diseases and/or in the improvement of anti-tumor treatments. The invention also relates to nucleic acids coding for the novel inhibitors. The invention relates further to the use of polypeptides, derived from these inhibitors in the treatment of NF-xcexaB related diseases and/or cancer. Furthermore, the invention concerns pharmaceutical preparations, comprising the novel inhibitors or the polypeptides, derived from these inhibitors.
NF-xcexaB is an ubiquitously expressed transcription factor that controls the expression of a diverse range of genes involved in inflammation, immune response, lymphoid differentiation, growth control and development. NF-xcexaB resides in the cytoplasm as an inactive dimer consisting of p50 and p65 subunits, bound to an inhibitory protein known as IxcexaB. The latter becomes phosphorylated and degraded in response to various environmental stimuli, such as pro-inflammatory cytokines, viruses, lipopolysaccharides, oxidants, UV light and ionizing radiation. This allows NF-xcexaB to translocate to the nucleus where it activates genes that play a key role in the regulation of inflammatory and immune responses, including genes that encode pro-inflammatory cytokines (IL-1xcex2, TNF, GM-CSF, IL-2, IL-6, IL-11, IL-17), chemokines (IL-8, RANTES, MIP-1xcex1, MCP-2), enzymes that generate mediators of inflammation (NO synthetase, cyclo-oxygenase), immune receptors (IL-2 receptor) and adhesion molecules (ICAM-1, VCAM-1, E-selectin). Some of these induced proteins can on their turn activate NF-xcexaB, leading to the further amplification and perpetuation of the inflammatory response. Recently, NF-xcexaB has been shown to have an anti-apoptotic role in certain cell types, most likely by inducing the expression of anti-apoptotic genes. This function may protect tumor cells against anti-cancer treatments and opens the possibility to use NF-xcexaB inhibiting compounds to sensitize the tumor cells and to improve the efficiency of the anti-cancer treatment.
Because of its direct role in regulating responses to inflammatory cytokines and endotoxin, activation of NF-xcexaB plays an important role in the development of different diseases such as (Barnes and Karin, 1997): chronic inflammatory diseases, i.e., rheumatoid arthritis, asthma and inflammatory bowel disease (Brand et al., 1996); acute diseases, i.e., septic shock (Remick, 1995); Alzheimer""s disease where the xcex2-amyloid protein activates NF-xcexaB (Behl et al., 1997); atherosclerosis, where NF-xcexaB may be activated by oxidized lipids (Brand et al., 1997); autoimmune diseases, i.e., such as systemic lupus erythematosis (Kaltschmidt et al., 1994); or cancer by up-regulating certain oncogenes or by preventing apoptosis (Luque et al., 1997). In addition, NF-xcexaB is also involved in viral infection since it is activated by different viral proteins, such as occurs upon infection with rhinovirus, influenza virus, Epstein-Barr virus, HTLV, cytomegalovirus or adenovirus. Furthermore, several viruses such as HIV have NF-xcexaB binding sites in their promoter/enhancer regions (Mosialos, 1997).
Because of the potential role of NF-xcexaB in many of the above mentioned diseases, NF-xcexaB and its regulators have drawn much interest as targets for the treatment of NF-xcexaB related diseases. Glucocorticoids are effective inhibitors of NF-xcexaB, but they have endocrine and metabolic side effects when given systematically (Barnes et al., 1993). Antioxidants may represent another class of NF-xcexaB inhibitors, but currently available antioxidants, such as acetyl-cysteine are relatively weak and unspecific (Schreck et al., 1991). Aspirin and sodium salicylate also inhibit activation of NF-xcexaB, but only at relatively high concentrations (Kopp and Gosh, 1994). There are some natural inhibitors of NF-xcexaB such as glyotoxin, derived from Aspergillus, but these compounds are too toxic to be used as a drug (Pahl et al., 1996). Finally, there may be endogenous inhibitors of NF-xcexaB, such as IL-10, that blocks NF-xcexaB through an effect on IxcexaB (Wang et al., 1995). However, total inhibition of NF-xcexaB in all cell types for prolonged periods is unwanted, because NF-xcexaB plays a crucial role in the immune response and other defensive responses.
An important role in the induction of NF-xcexaB by TNF and IL1 has recently been demonstrated for TNF-receptor associated factors, TRAF2 and TRAF6, which are recruited to the stimulated TNF-receptor and IL-1 receptor, respectively (Rothe et al., 1995; Cao et al., 1996). Over expression of TRAF2 or TRAF6 activates NF-xcexaB, whereas dominant negative mutants inhibit TNF or IL-1 induced activation of NF-xcexaB in most cell types. TRAF2 knock out studies have recently shown that TRAF2 is not absolutely required for NF-xcexaB activation, presumably because of redundancy within the TRAF family (Yeh et al. 1997). The TRAF induced signaling pathway to NF-xcexaB was further resolved by the identification of the TRAF-interacting protein NIK, which mediates NF-xcexaB activation upon TNF and IL-1 stimulation by association and activation of IxcexaB kinase-xcex1 and -xcex2 (IKK) (Malinin et al, 1997; Regnier et al., 1997; DiDonato et al., 1997; Zandi et al., 1997; Woronicz et al., 1997). The latter are part of a large multi-protein NF-xcexaB activation complex and are responsible for phosphorylation of IxcexaB, leading to its subsequent degradation and to translocation of released, active NF-xcexaB to the nucleus. This allows a more specific inhibition of NF-xcexaB activation by stimuli (including TNF and IL-1) that activate TRAF pathways. Based on this principle, WO 97/37016 discloses the use of NIK and other TRAF interacting proteins for the modulation of NF-xcexaB activity.
Another protein that can associate with TRAF2 is the zinc finger protein A20 (Song et al., 1996). The latter is encoded by an immediate early response gene induced in different cell lines upon stimulation by TNF or IL-1 (Dixit et al, 1990). Interestingly, over expression of A20 blocks both TNF and IL-1 induced NF-xcexaB activation (Jaattela et al., 1996). However, the mechanism by which A20 blocks NF-xcexaB activation is totally unknown. In contrast to NIK, A20 does not seem to act directly on IxcexaB resulting in alternative pathway to modulate NF-xcexaB activation.
De Valck et al. (1997) isolated an A20 binding protein, so-called 14-3-3, using a yeast two-hybrid assay and demonstrated that NF-xcexaB inhibition was independent from the binding of A20 to 14-3-3.
It is shown herein that other new A20 interacting proteins unexpectedly can modulate and/or inhibit NF-xcexaB activation.
The invention includes an isolated functional protein comprising an amino acid sequence with 70-100% homology to the amino acid sequence depicted in SEQ. ID. NO. 2, or comprising an amino acid sequence with 70-100% homology to the amino acid sequence depicted in SEQ. ID. NO. 3, or, in the alternative, comprising an amino acid sequence with 70-100% homology to the amino acid sequence depicted in SEQ. ID. NO. 5.
More specifically, the functional protein comprises an amino acid sequence with 70-100% homology to the amino acids 54-647 of SEQ. ID. NO. 2, even more specifically the functional protein comprises an amino acid sequence with 70-100% homology to the amino acids 390-647 of SEQ. ID. NO. 2, or, in the alternative, and/or comprising an amino acid sequence with 70-100% homology to the amino acids 420-647 of SEQ. ID. NO.2.
Homology, in this context, means identical or similar to the referenced sequence, while obvious replacements/modifications of any of the amino acids provided, are included as well. A homology search in this respect can be performed with the BLAST-P (Basic Local Alignment Search Tool) program well known to a person skilled in the art. For the corresponding nucleic acid sequence homology is referred to the BLASTX and BLASTN programs known in the art.
One aspect of the invention is to offer novel modulators and/or inhibitors of TNF and/or IL-1 induced NF-xcexaB activation pathways.
An important embodiment of the invention is a protein comprising at least the amino acids of SEQ. ID. NO. 2.
Another embodiment of the invention is a protein comprising at least the amino acids 54-647 of SEQ. ID. NO. 2, as represented in SEQ. ID. NO. 3.
A further embodiment of the invention is a protein comprising at least the amino acids of SEQ ID. NO.5.
A further aspect of the invention is the use of protein comprising the amino acids 420-647 of SEQ ID. NO. 2 to modulate and/or inhibit the NF-xcexaB related pathway, especially the TNF and/or IL-1 induced pathways.
In addition, the invention concerns the use of a protein, comprising the consensus sequence shown in SEQ ID NO: 8 and/or SEQ ID NO: 9, to modulate and/or inhibit the TNF and/or IL-1 induced, NF-xcexaB related pathway.
Another aspect of the invention is the use of these proteins in a screening method to screen compounds that interfere with the interaction of these protein(s) with other protein components of the NF-xcexaB related pathway.
Another embodiment of the invention is the use of the above mentioned proteins, or the use of protein components screened by the above mentioned method to sensitize tumor cells and/or improve the anti-cancer treatment.
In the alternative, the present invention relates to a method for identifying and obtaining an activator or inhibitor of A20 interacting protein(s) comprising the steps of:
(a) combining a compound to be screened with a reaction mixture containing the protein of the invention and a read out system capable of interacting with the protein under suitable conditions;
(b) maintaining the reaction mixture in the presence of the compound or a sample comprising a plurality of compounds under conditions which permit interaction of the protein with the read out system;
(c) identifying or verifying a sample and compound, respectively, which leads to suppression or activation of the read out system.
The term xe2x80x9cread out systemxe2x80x9d in context with the present invention means a DNA sequence which upon transcription and/or expression in a cell, tissue or organism provides for a scorable and/or selectable phenotype. Such read out systems are well known to those skilled in the art and comprise, for example, recombinant DNA molecules and marker genes as described above.
The term xe2x80x9cplurality of compoundsxe2x80x9d in a method of the invention is understood as a plurality of substances which may be identical.
The compound or plurality of compounds may be comprised in, for example, samples, e.g., cell extracts from animals or microorganisms. Furthermore, the compound(s) may be known in the art but hitherto not known to be capable of suppressing or activating A20 interacting proteins. The reaction mixture may be a cell free extract or may comprise a cell or tissue culture. Suitable set ups for the method of the invention are known to the person skilled in the art and are, for example, generally described in Alberts et al., Molecular Biology of the Cell, (3rd ed. 1994). The plurality of compounds may be, for instance, added to the reaction mixture or culture medium, or may be injected into the cell.
If a sample containing a compound or a plurality of compounds is identified in the method of the invention, then it is possible to isolate the compound from the original sample identified as containing the compound capable of suppressing or activating A20 interacting proteins. Additionally, one can further subdivide the original sample, for example, if it consists of a plurality of different compounds, so as to reduce the number of different substances per sample. The method can then be repeated with the subdivisions of the original sample. Depending on the complexity of the samples, the steps described above can be performed several times, preferably until the sample identified according to the method of the invention only comprises a limited number of, or only one substance(s). Preferably, the sample comprises substances of similar chemical and/or physical properties, and most preferably the substances are identical. The compounds which can be tested and identified according to a method of the invention may be expression libraries, e.g., cDNA expression libraries, peptides, proteins, nucleic acids, antibodies, small organic compounds, hormones, peptidomimetics, PNAs or the like (Milner, Nature Medicine 1 (1995), 879-880; Hupp, Cell 83 (1995), 237-245; Gibbs, Cell 79 (1994), 193-198 and references cited supra).
In the alternative, the invention also relates to a DNA sequence encoding the referenced proteins or a DNA sequence encoding an immunologically active and/or functional fragment of such a protein, selected from the group consisting of:
(a) DNA sequences comprising a nucleotide sequence encoding a protein comprising the amino acid sequence as given in SEQ ID NO: 2;
(b) DNA sequences comprising a nucleotide sequence as given in SEQ ID NO: 1;
(c) DNA sequences hybridizing with the complementary strand of a DNA sequence as defined in (a) or (b) and encoding an amino acid sequence which is at least 70% identical to the amino acid sequence encoded by the DNA sequence of (a) or (b);
(d) DNA sequences, the nucleotide sequence of which is degenerated as a result of the genetic code to a nucleotide sequence of a DNA sequence as defined in any one of (a) to (c); and
(e) DNA sequences encoding a fragment of a protein encoded by a DNA sequence of any one of (a) to (d).
Thus, the invention consists of DNA molecules, also called nucleic acid sequences, encoded for the above mentioned proteins preferably a nucleic acid sequence, with 70-100% homology to the DNA sequence depicted in SEQ. ID. NO. 1, and/or a nucleic acid sequence with 70-100% homology to the DNA sequence depicted in SEQ. ID. NO. 4.
Homology in this context means that the respective nucleic acid molecules or encoded proteins are functionally and/or structurally equivalent. The nucleic acid molecules that are homologous to the nucleic acid molecules described above and that are derivatives of the nucleic acid molecules are, for example, variations of the nucleic acid molecules which represent modifications having the same biological function. In particular, the modifications encode proteins with the same or substantially the same biological function. They may be naturally occurring variations, such as sequences from other varieties or species, or mutations. These mutations may occur naturally or may be obtained by mutagenesis techniques. The allelic variations may be naturally occurring allelic variants as well as synthetically produced or genetically engineered variants.
The proteins encoded by the various derivatives and variants of the above-described nucleic acid molecules have similar common characteristics, such as biological activity, molecular weight, immunological reactivity, conformation, etc., as well as physical properties, such as electrophoretic mobility, chromatographic behavior, sedimentation coefficients, pH optimum, temperature optimum, stability, solubility, spectroscopic properties, etc.
The present invention also relates to vectors, particularly plasmids, cosmids, viruses, bacteriophages and other vectors used conventionally in genetic engineering that contain a nucleic acid molecule according to the invention. Methods which are well known to those skilled in the art can be used to construct various plasmids and vectors; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.
Alternatively, the nucleic acid molecules and vectors of the invention can be reconstituted into liposomes for delivery to target cells.
In a preferred embodiment, the nucleic acid molecule present in the vector is operably linked to (a) control sequence(s) which allow the expression of the nucleic acid molecule in prokaryotic and/or eukaryotic cells.
The term xe2x80x9ccontrol sequencexe2x80x9d refers to regulatory DNA sequences which are necessary to affect the expression of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism. In procaryotes, control sequences generally include promoter, ribosomal binding site, and terminators. In eucaryotes, control sequences generally include promoters, terminators and, in some instances, enhancers, transactivators or transcription factors. The term xe2x80x9ccontrol sequencexe2x80x9d is intended to include, at a minimum, all components that are necessary for expression, and may also include additional advantageous components.
The term xe2x80x9coperably linkedxe2x80x9d refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence xe2x80x9coperably linkedxe2x80x9d to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. In case the control sequence is a promoter, it is obvious for a skilled person that double-stranded nucleic acid is used.
Thus, the vector of the invention is preferably an expression vector. An xe2x80x9cexpression vectorxe2x80x9d is a construct that can be used to transform a selected host cell and provides for expression of a coding sequence in the selected host. Expression vectors can, for instance, be cloning vectors, binary vectors or integrating vectors. Expression comprises transcription of the nucleic acid molecule preferably into a translatable mRNA. Regulatory elements ensuring expression in prokaryotic and/or eukaryotic cells are well known to those skilled in the art.
The present invention furthermore relates to host cells comprising a vector as described above or a nucleic acid molecule according to the invention wherein the nucleic acid molecule is foreign to the host cell.
By xe2x80x9cforeignxe2x80x9d is meant that the nucleic acid molecule is either heterologous or homologous with respect to the host cell. xe2x80x9cHeterologousxe2x80x9d means derived from a cell or organism with a different genomic background. xe2x80x9cHomologousxe2x80x9d means located in a different genomic environment than the naturally occurring counterpart of the nucleic acid molecule. Thus, if the nucleic acid molecule is homologous with respect to the host cell, it is not located in its natural location in the genome of the host cell, but it is surrounded by different genes. In this case, the nucleic acid molecule may be either under the control of its own promoter or under the control of a heterologous promoter. The vector or nucleic acid molecule, according to the invention, which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained in some form extra-chromosomally. It is also possible that the nucleic acid molecule of the invention can be used to restore or create a mutant gene via homologous recombination (Paszkowski (ed.), Homologous Recombination and Gene Silencing in Plants, (Kluwer Academic Publishers 1994)).
The host cell can be any prokaryotic or eukaryotic cell, such as bacterial, insect, fungal, plant or animal cells. Preferred fungal cells are, for example, those of the genus Saccharomyces, in particular those of the species S. cerevisiae. 
The invention also includes a method for preparing A20 interacting proteins which method comprises the cultivation of host cells that due to the presence of a vector or a nucleic acid molecule according to the invention, are able to express such a protein under conditions which allow expression of the protein and thus recovery of the so-produced protein from the culture.
The term xe2x80x9cexpressionxe2x80x9d means the production of a protein or nucleotide sequence in the cell. However, the term also includes expression of the protein in a cell-free system. It includes transcription into an RNA product, post-transcriptional modification and/or translation to a protein product or polypeptide from a DNA encoding that product, as well as possible post-translational modifications. Depending on the specific constructs and conditions used, the protein may be recovered from the cells, from the culture medium or from both. For the person skilled in the art, it is well known that it is not only possible to express a native protein, but also to express the protein as fusion polypeptides or to add signal sequences directing the protein to specific compartments of the host cell, for example, ensuring secretion of the peptide into the culture medium, etc. Furthermore, such a protein and fragments thereof can be chemically synthesized and/or modified according to standard methods.
The terms xe2x80x9cproteinxe2x80x9d and xe2x80x9cpolypeptidexe2x80x9d used in this application are interchangeable. xe2x80x9cPolypeptidexe2x80x9d refers to a polymer of amino acids (amino acid sequence) and does not refer to a specific length of the molecule. Thus peptides and oligopeptides are included within the definition of polypeptide. This term also refers to and includes post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
The present invention furthermore relates to proteins encoded by the nucleic acid molecules according to the invention or produced or obtained by the above-described methods, and to functional and/or immunologically active fragments of such A20 interacting proteins. The proteins and polypeptides of the present invention are not necessarily translated from a designated nucleic acid sequence. The polypeptides may be generated in any manner, including for example, chemical synthesis, or expression of a recombinant expression system, or isolation from a suitable viral system. The polypeptides may include one or more analogs of amino acids, phosphorylated amino acids or unnatural amino acids. Methods of inserting analogs of amino acids into a sequence are known in the art. The polypeptides may also include one or more labels, which are known to those skilled in the art. In this context, it is also understood that the proteins according to the invention may be further modified by conventional methods known in the art. By providing the proteins according to the present invention it is also possible to determine fragments which retain biological activity, namely the mature, processed form. This allows the construction of chimeric proteins and peptides comprising an amino sequence derived from the protein of the invention, which is crucial for its binding activity. The other functional amino acid sequences may be either physically linked by, for example, chemical means to the proteins of the invention or may be fused by recombinant DNA techniques well known in the art.
The term xe2x80x9cfunctional fragment of a sequencexe2x80x9d or xe2x80x9cfunctional part of a sequencexe2x80x9d means a truncated sequence of the original sequence referred to. The truncated sequence (nucleic acid or protein sequence) can vary widely in length; the minimum size being a sequence of sufficient size to provide a sequence with at least a comparable function and/or activity of the original sequence referred to, while the maximum size is not critical. In some applications, the maximum size usually is not substantially greater than that required to provide the desired activity and/or function(s) of the original sequence. Typically, the truncated amino acid sequence will range from about 5 to about 60 amino acids in length. More typically, however, the sequence will be a maximum of about 50 amino acids in length, preferably a maximum of about 30 amino acids. It is desirable to select sequences of at least about 10, 12 or 15 amino acids, up to a maximum of about 20 or 25 amino acids.
Furthermore, folding simulations and computer redesign of structural motifs of the protein of the invention can be performed using appropriate computer programs (Olszewski, Proteins 25 (1996), 286-299; Hoffman, Comput. Appl. Biosci. 11 (1995), 675-679). Computer modeling of protein folding can be used for the conformational and energetic analysis of detailed peptide and protein models (Monge, J. Mol. Biol. 247 (1995), 995-1012; Renouf, Adv. Exp. Med. Biol. 376 (1995), 37-45). In particular, the appropriate programs can be used for the identification of interactive sites of the inventive protein, its receptor, its ligand or other interacting proteins by computer assistant searches for complementary peptide sequences (Fassina, Immunomethods 5 (1994), 114-120. Further appropriate computer systems for the design of protein and peptides are described in the prior art, for example in Berry, Biochem. Soc. Trans. 22 (1994),1033-1036; Wodak, Ann. N. Y. Acad. Sci. 501 (1987), 1-13; Pabo, Biochemistry 25 (1986), 5987-5991. The results obtained from the above-described computer analysis can be used for, e.g., the preparation of peptidomimetics of the protein of the invention or fragments thereof. Such pseudopeptide analogues of the natural amino acid sequence of the protein may very efficiently mimic the parent protein (Benkirane, J. Biol. Chem. 271 (1996), 33218-33224). For example, incorporation of easily available achiral amino acid residues into a protein of the invention or a fragment thereof results in the substitution of amide bonds by polymethylene units of an aliphatic chain, thereby providing a convenient strategy for constructing a peptidomimetic (Banerjee, Biopolymers 39 (1996), 769-777). Superactive peptidomimetic analogues of small peptide hormones in other systems are described in the prior art (Zhang, Biochem. Biophys. Res. Commun. 224 (1996), 327-331). Appropriate peptidomimetics of the protein of the present invention can also be identified by the synthesis of peptidomimetic combinatorial libraries through successive amide alkylation and testing the resulting compounds, e.g., for their binding and immunological properties. Methods for the generation and use of peptidomimetic combinatorial libraries are described in the prior art. See, e.g., Ostresh, Methods in Enzymology 267 (1996), 220-234; Dorner, Bioorg. Med. Chem. 4 (1996), 709-715.
Furthermore, a three-dimensional and/or crystallographic structure of the protein of the invention can be used for the design of peptidomimetic inhibitors of the biological activity of the protein of the invention (Rose, Biochemistry 35 (1996), 12933-12944; Rutenber, Bioorg. Med. Chem. 4 (1996), 1545-1558).
Furthermore, the present invention relates to antibodies specifically recognizing a A20 interacting protein according to the invention or parts, i.e. specific fragments or epitopes, of such a protein. The antibodies of the invention can be used to identify and isolate other A20 interacting proteins and genes in any organism. These antibodies can be monoclonal antibodies, polyclonal antibodies or synthetic antibodies as well as fragments of antibodies, such as Fab, Fv or scFv fragments etc. Monoclonal antibodies can be prepared, for example,, by the techniques as originally described in Kohler and Milstein, Nature 256 (1975), 495, and Galfre, Meth. Enzymol. 73 (1981), 3, which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals. Furthermore, antibodies or fragments thereof to the aforementioned peptides can be obtained by using methods which are described, for example, in Harlow and Lane xe2x80x9cAntibodies, A Laboratory Manualxe2x80x9d, CSH Press, Cold Spring Harbor, 1988. These antibodies can be used, for example, for the immunoprecipitation and immunolocalization of proteins according to the invention. Additionally, the antibodies can be used for the monitoring of the synthesis of such proteins, for example, in recombinant organisms, and for the identification of compounds interacting with the protein according to the invention. For example, surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies selections, yielding a high increment of affinity from a single library of phage antibodies which bind to an epitope of the protein of the invention (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13). In many cases, the binding phenomena of antibodies to antigens is equivalent to other ligand/anti-ligand binding.
The invention also relates to a diagnostic composition comprising at least one of the aforementioned nucleic acid molecules, vectors, proteins, antibodies or compounds and optionally suitable means for detection.
The diagnostic compositions may be used for methods for detecting expression of related A20 interacting proteins. This is accomplished by detecting the presence of the corresponding mRNA which comprises isolation of mRNA from a cell, contacting the mRNA obtained with a probe comprising a nucleic acid probe as described above under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and detecting the expression of the protein in the cell. Further methods of detecting the presence of a protein according to the present invention comprises immunotechniques well known in the art, for example, enzyme-linked immunosorbent assays.
The invention also relates to a pharmaceutical composition comprising one or more compounds, obtained by the above mentioned screening method, in a biologically active amount, for the treatment of NF-xcexaB related diseases such as respiratory disorders, particularly adult respiratory distress syndrome, allograft rejection, chronic inflammatory diseases such as rheumatoid arthritis, asthma or inflammatory bowel disease, and/or autoimmune diseases such as systemic lupus erythematosis.
In another aspect, the invention relates to a pharmaceutical composition comprising one or more of the above mentioned proteins in a biologically active amount, for the treatment of NF-xcexaB related diseases such as respiratory disorders, particularly adult respiratory distress syndrome, allograft rejection, chronic inflammatory diseases such as rheumatoid arthritis, asthma or inflammatory bowel disease, and/or autoimmune diseases such as systemic lupus erythematosis.
The invention also concerns a pharmaceutical composition comprising one or more of the above mentioned proteins and/or one or more of the above mentioned compounds in a biologically active amount, for a treatment to sensitize tumor cells.